EP1855102B1

EP1855102B1 - Fluorescence detecting device and fluorescence detecting method

Info

Publication number: EP1855102B1
Application number: EP06713774A
Authority: EP
Inventors: N. c/o Mitsui Eng. & Shipbldg. Co. Ltd. KIMURA; K. c/o Mitsui Eng. & Shipbldg. Co. Ltd. DOI; T. c/o Mitsui Eng. & Shipbldg. Co. Ltd. YUMII; T. c/o Mitsui Eng. & Shipbldg. Co. Ltd. YOSHIDA; S. c/o Mitsui Eng. & Shipbldg. Co. Ltd. NAKADA; H. c/o Mitsui Eng. & Shipbldg. Co. Ltd. HAYASHI; K. c/o Mitsui Eng & Shipbldg Co. Ltd HOSHISHIMA
Original assignee: Mitsui Engineering and Shipbuilding Co Ltd
Current assignee: Mitsui Engineering and Shipbuilding Co Ltd
Priority date: 2005-02-15
Filing date: 2006-02-15
Publication date: 2011-09-07
Anticipated expiration: 2026-02-15
Also published as: US20090012721A1; DK1855102T3; EP2369326A1; US7822558B2; EP1855102A1; EP1855102B8; EP1855102A4; EP2369326B1; WO2006088047A1

Abstract

A fluorescence detecting device which irradiates an object to be measured with a laser beam, receives florescence emitted from the object, and processes the florescence signal thus generated . The fluorescence signal detecting device comprises a laser light source part for emitting a laser beam with which the object is irradiated, a light receiving part for outputting a fluorescence signal of the fluorescence emitted from the object irradiated with the laser beam, a light source control part for generating a modulation signal of a predetermined frequency with which the intensity of the laser beam is time-modulated, and a processing part for computing the fluorescence relaxation time of the fluorescence emitted from the object by using the modulation signal from the fluorescence signal outputted from the light-receiving part when the object is irradiated with the time-modulated laser beam. From the detection values including the phase information on the fluorescence emitted from a labeled sample and collected by this device, the intensity of the fluorescence emitted from the labeled sample is determined.

Description

Technical Field

The present invention relates to a fluorescence detecting device and a fluorescence detecting method, in which an object to be measured is irradiated with a laser beam that has been subjected to intensity modulation, a fluorescence signal from the object to be measured which is attributable to the irradiation is received, and signal processing is conducted on the signal. The fluorescence detecting device and the fluorescence detecting method are applied to, for example, an analyzing device, such as a flow cytometer used in a medical or biological field, which is capable of analyzing the object to be measured in a short period of time by identifying fluorescence that is emitted by a fluorochrome due to irradiation of the laser beam to thereby identify the object to be measured while the object to be measured is flowing in a sheath solution. More particularly, the present invention relates to a fluorescence detecting device and a fluorescence detecting method, in which respective fluorescence intensities are obtained by using fluorescence detected values of plural labeled samples, the fluorescence detected values being generated by irradiating the plural labeled samples with the laser beam at the same time by using the flow cytometer or the like.

Background Art

A fluorescence detecting device that receives fluorescence from the fluorochrome of the object to be measured by irradiating the object to be measured with the laser beam to identify the type of the object to be measured is employed in the flow cytometer used in the medical or biological field.
Specifically, the flow cytometer is a device that mixes samples such as cells, DNAs, RNAs, enzymes, proteins, or micro beads, which are labeled as the fluorochrome, with a normal saline to produce a sample solution by using the binding of antigen-antibody reaction and the like. Then, the device allows the sample solution to flow so as to be encompassed with another solution called "sheath solution", resulting that a laminar sheath flow in which the labeled samples flow at a speed of about 10 m or lower per second is formed while a pressure is exerted on the laminar sheath flow. The device irradiates the sheath flow with a laser beam to measure the fluorescence or a scattered light in each of the samples. For example, in the case where a variety of samples are to be analyzed, the flow cytometer measures the fluorescent intensity of the fluorescence that is generated by the sample, and identifies which sample having which fluorescence characteristics has passed by among the many kinds of samples. In this case, labeled samples to which fluorochromes of different kinds are adhered are used for the many kinds of samples. The flow cytometer irradiates the labeled sample with the laser beam, and measures the fluorescence that is generated with the irradiation of the laser beam.
Further, the flow cytometer is capable of measuring an intracellular relative quantity of, for example, DNAs, RNAs, enzymes, or proteins within a cell, and also analyzing activity thereof in a short time. Further, there is used a cell sorter that specifies a specific type of cell or chromosome by fluorescence, and selectively collects only the specific cell or chromosome in a live state in a short time.
In the use of the cell sorter, it is required to specify more objects to be measured from information on fluorescence in a short time.
In the following Non-patent Document 1, there is disclosed a flow cytometer that irradiates fluorochromes with plural laser beams that are different in the wavelength band such as 488 nm, 595 nm, and 633 nm, separates plural kinds of fluorescence that are different in the wavelength band which are generated from the fluorochrome by the respective laser beams by using a band pass filter, and detects the separated fluorescence by means of a photoelectron multiple tube (PMT). With the above configuration, it is possible to identify the fluorescence from the plural fluorescent reagents (fluorochrome) to specify the plural kinds of objects to be measured at once.
However, although the wavelength range of the fluorescence that is generated from the fluorescent reagent has a relatively wide width such as about 400 to 800 nm, only 3 to 4 wavelength bands of the fluorescent reagent can be effectively used for an identifiable labels in the visible wavelength range. An increase in the number of fluorescence that can be identified by employing the plural fluorescent reagents is restricted.
Further, in order to increase the number of identifiable fluorescence, it is possible to increase the number of identifiable fluorescence by using the wavelength of fluorescence as well as the intensity of detected fluorescence. However, even in this case, the number of identifiable fluorescence by using the intensity of fluorescence is about 2 to 5, and even if this number is combined with the above-mentioned number of identifiable fluorescence of about 3 to 4 wavelength bands, the number of identifiable fluorescence is about 20 at maximum. For that reason, there arises such a problem that it is difficult to identify and analyze an extremely large number of objects to be measured in a short time even if the above flow cytometer is used.
For example, in the case where a biologic material such as DNA is analyzed by the flow cytometer, fluorochrome is adhered to the biologic material with a fluorescent reagent in advance, and the biologic material is labeled by a fluorochrome different from fluorochromes that have been adhered to micro beads which will be described later. Then, the biologic material is mixed with a solution containing micro beads of 5 to 20 µm in diameter, the micro beads having a surface provided with a unique structure such as carboxyl group. The structure of carboxyl group acts on a biologic material of a certain known structure, and conducts a biological coupling therewith. Accordingly, in the case where the flow cytometer detects fluorescence from the micro beads and fluorescence of the biologic material at the same time, it is found that the biologic material is biologically coupled with the structure of the micro beads. As a result, it is possible to analyze the characteristic of the biologic material. However, in order to analyze the characteristics of the biologic material in a short time by providing various micro beads having various coupling structures, an extremely large number of kinds of fluorochromes are required. However, since the number of kinds of fluorescent reagents identifiable at the same time is small, it is impossible to analyze the biologic material efficiently in a short time by using a large variety of micro beads at once.
Further, there is proposed a method in which plural measurement points at which an object to be measure is irradiated with laser beams to measure fluorescence are provided in the longitudinal direction of a flow tube, and the laser beams irradiated at the respective measurement points are prevented from interfering with each other. However, in this case, it is necessary to provide a large number of laser beams and a large number of light receiving sections in correspondence with the number of measurement points. Further, since a flow tube that forms the flow cell is elongated, the flow path resistance of the sheath solution that flows in the tube becomes large, and a pressure to be exerted on the sheath solution becomes large. For that reason, there arises such a problem that the device is increased in size.
Non-patent Document 1 : http://www.bddbiosciences. com/pharmingen/protocols/Fluorochrome_Absorption.shtml (searched on January 23, 2005)
A fluorescence detecting device is disclosed in US 5 270 548A patent. This device may be considered as the closest state of the art. An instrument for analyzing a multiplicity of fluorescent dyes is disclosed in US 2004/119974 A1 application. Finally, Keij JF et al. disclose in their article "Flow cytometric characterization and classification of multiple dual-color fluorescent microspheres using fluorescence lifetime" cytometry, Alan Liss, New-York, US LNKD-DOI:10.1002/ (SICI) 1097-0320 (19981101) 33:3 318::AID-CYTO5 3.0.CO;2-C, vol. 33, n° 3, 1 January 1998 (1998-01-01), pages 318-323, XP0008635701SSN: 0196-4763, a flow cytometry process.

Disclosure of the Invention

Problems to be solved by the Invention

Under the circumstances, to solve the above problems, it is an object of the present invention to provide a fluorescence detecting device which is capable of identifying the kinds of fluorescence that is generated from a large number of samples that are objects to be measured such as micro beads through signal processing. In particular, the device is capable of identifying fluorescence signals efficiently in a short time, when the fluorescence detecting device irradiates the objects to be measured with a laser beam and receives fluorescence signals from the objects to be measured to conduct signal processing. For example, the fluorescence detecting device is preferably used in a flow cytometer.

Means for solving the Problems

The present invention provides a fluorescence detecting device using an intensity-modulated laser beam, which irradiates an object to be measured with a laser beam to receive fluorescence generated by the object to be measured, and carries out signal processing of a fluorescence signal obtained when receiving the fluorescence, comprising: a laser light source section that outputs the laser beam with which the object to be measured is irradiated; a light receiving section that outputs the fluorescence signal of the fluorescence generated by the object to be measured which is irradiated with the laser beam; a light source control section that generates a modulation signal having a given frequency in order to time-modulate an intensity of the laser beam that is output from the laser light source section; and a processing section that calculates, by using the modulation signal, a fluorescence relaxation time of the fluorescence of the object to be measured based on the fluorescence signal that is output from the light receiving section by irradiating the object to be measured with the time-modulated laser beam.
In the invention, the processing section preferably obtains a phase delay with respect to the modulation signal of the fluorescence signal to calculate the fluorescence relaxation time.
Preferably, the light source control section uses as a pulse control signal a coding sequence signal that is selected from a plurality of coding sequence signals which have signal values of one bit coded with a given length and are orthogonal to each other, and sets and controls on/off of output of the laser beam so that an on-time of the output of the laser beam from the laser light source section is longer than one cycle time of the time modulation of the laser beam, and the processing section calculates the fluorescence relaxation time and identifies the fluorescence from the object to be measured, by using the coding sequence signal based on a light receiving signal that is output from the light receiving section. Here, the plurality of coding sequence signals are preferably configured by shifting one coding sequence signal in a bit direction and the coding sequence signals become orthogonal to each other by the shifting.
Preferably, the laser light source section includes a plurality of laser light sources that output a plurality of laser beams, wherein the light source control section controls the on/off of outputs of the laser beams from the plurality of laser light sources by using the plurality of coding sequence signals that are orthogonal to each other, and wherein the processing section separates each of fluorescence signals of the fluorescence that is generated by the object to be measured by irradiation of the respective laser beams, from the fluorescence signals which are overlapped together and outputted from the light receiving section, including optical signals of the plurality of laser beams, by using the coding sequence signals used for the outputs of the laser beams.

Effects of the Invention

According to the present invention, micro beads etc. are set as objects to be measured, and the objects to be measured are irradiated with a laser beam that has been modulated in intensity at a given frequency to obtain a fluorescence relaxation time of the fluorescence that is generated by the irradiation with the laser beam. Since the fluorescence relaxation time varies depending on the kind of fluorochromes, it is possible to identify the kinds of fluorescence, particularly the kinds of objects to be measured by using the fluorescence relaxation time. In other words, since the fluorescence relaxation time can be used to identify the fluorescence besides the wavelength of fluorescence and the intensity of fluorescence which have been conventionally used to identify the fluorescence, the number of identifiable kinds of fluorescence is increased. In particular, the present invention is effective for a flow cytometer that efficiently specifies the object to be measured in a short time by using a large number of fluorochromes.
Further, in the case of using plural laser beams, coding sequence signals that are orthogonal to each other in each of the laser beams are used as pulse control signals of the laser beams, thereby making it possible to specify which laser beam causes the received fluorescence signal to be genarated by the irradiation. As a result, it is possible to efficiently specify the objects to be measured in a short time.

Brief Description of the Drawings

Fig. 1 is a schematic structural diagram showing a flow cytometer using a fluorescence detecting device that uses an intensity-modulated laser beam according to the present invention.
Fig. 2 is a schematic structural diagram showing an example of laser light sources that are used in the fluorescence detecting device outputting an intensity-modulated laser beam according to the present invention.
Figs. 3 are graphs each schematically showing a spectrum intensity distribution of a laser beam that is output from the laser light sources shown in Fig. 2 and light that is generated by a fluorochrome.
Fig. 4 is a schematic structural diagram showing an example of a light receiving section that is used in the fluorescence detecting device outputting an intensity-modulated laser beam according to the present invention.
Fig. 5 is a schematic structural diagram showing an example of a control/processing section that is used in the fluorescence detecting device outputting an intensity-modulated laser beam according to the present invention.
Fig. 6 is a diagram for explaining an IQ mixer in the control/processing section shown in Fig. 5.
Figs. 7A and 7B are diagrams showing an example of respective signals that are generated by the fluorescence detecting device outputting an intensity-modulated laser beam according to the present invention.
Fig. 8 is a diagram for explaining characteristics of a fluorescence intensity of light that is generated by the fluorochrome.

Description of Symbols

10, flow cytometer
12 specimen
20, signal processing device
22, laser light source section
22r R light source
22g G light source
22b B light source
23a₁, 23a₂, 26b₁, 26b₂ dichroic mirror
23c, 26a, lens system
24, 26, 124, light receiving section
26c₁, 26c₂, 26c₃, band pass filter
27a-27c, photoelectric converter
28, control/processing section
30, tube
32, collecting vessel
34r, 34g, 34b laser driver
35, 48, 56 power splitter
40, signal generating section
42, signal processing section
44, controller
46, oscillator
50, 52, 54a, 54b, 54c, 64, 150, 152, amplifier
58a, 58b, 58c IQ mixer
60, system controller
62, low pass filter
66, A/D converter
80, analyzing device

Best Mode for carrying out the Invention

Hereinafter, a description will be given in more detail of a flow cytometer that preferably uses a fluorescence detecting device and a fluorescence detecting method using an intensity-modulated laser beam according to the present invention.
Fig. 1 is a schematic structural diagram showing a flow cytometer 10 using a fluorescence detecting device using an intensity-modulated laser beam according to the present invention.
A flow cytometer 10 includes a signal processing device 20 that irradiates a specimen 12 such as micro beads to be measured with a laser beam, and detects a flueroscence signal of flueroscence that is generated by fluorochromes disposed in the specimen 12 to process a signal, and an analyzing device (computer) 80 that analyzes the objects to be measured in the specimen 12 on the basis of the processed results that have been obtained by the signal processing device 20.
The signal processing device 20 includes a laser light source section 22, light receiving sections 24 and 26, a control/processing section 28 including a control section that modulates in intensity the laser beam from the laser light source section 22 at a given frequency and controls the on/off operation of output of the laser beam, and a signal processing section that identifies a fluorescence signal from the specimen 12, and a tube 30 that allows the specimen 12 included in a sheath solution which forms a high speed flow, thereby forming a flow cell.
A collecting vessel 32 is disposed at the outlet of the tube 30. A cell sorter for separating biologic material such as specific cells in the specimen 12 within a short time after the irradiation of the laser beam may be disposed in the flow cytometer 10 so as to separate the biologic material in different collecting vessels.
The laser light source section 22 is a section that outputs three laser beams different in wavelength, for example, laser beams of λ₁=405 nm, λ₂=533 nm, and λ₃= 650 nm. A lens system is disposed in such a manner that the laser beam is focused to a given position within the tube 30. A measurement point of the specimen 12 is defined at the focus position of the laser beam.
Fig. 2 is a diagram showing an example of the configuration of the laser light source section 22.
The laser light source section 22 is a section that outputs the laser beam having a wavelength of a vissible band of 350 nm to 800 nm, modulated in intensity and modulated in coding.
The laser light source section 22 has an R light source 22r, a G light source 22g, and a B light source 22b. The R light source 22r mainly emits a red laser beam R as a CW (continuous wave) laser beam that is constant in intensity, and also intermittently outputs the laser beam R while modulating the intensity of the CW laser beam at a given frequency. The G light source 22g emits a green laser beam G as a CW laser beam that is constant in intensity, and also intermittently outputs the laser beam G while modulating the intensity of the CW laser beam at a given frequency. The B light source 22b emits a blue laser beam B as a CW laser beam that is constant in intensity, and also intermittently outputs the laser beam B while modulating the intensity of the CW laser beam at a given frequency.
Further, the laser light source section 22 includes dichroic mirrors 23a₁, 23a₂, a lens system 23c that focuses a laser beam consisting of the laser beams R, G, and B on a measurement point in the tube 30, laser drivers 34r, 34g, and 34b that drive the R light source 22r, the G light source 22g, and the B light source 22b, respectively, and a power splitter 35 that distributes the supplied signal to the laser drivers 34r, 34g, and 34b, respectively.
The laser light sources that output those laser beams can be formed of, for example, a semiconductor laser. The laser beam has an output of, for example, about 5 to 100 mW. On the other hand, the frequency (modulation frequency) that modulates the intensity of the laser beam, which is slightly longer in the cycle time than the fluorescence relaxation time is, for example, 10 to 100 MHz.
The dichroic mirror 23a₁ is a mirror that transmits the laser beam R and reflects the laser beam G, and the dichroic mirror 23a₂ is a mirror that transmits the laser beams R and G, and reflects the laser beam B.
With the above configuration, the laser beams R, G, and B are combined together into an irradiation beam with which the specimen 12 is irradiated at the measurement point.
The laser drivers 34r, 34g, and 34b are connected to the control/processing section 28, and configured in such a manner that the intensity of the output of the laser beams R, G, and B, and the on/off operation of the output thereof are controlled. In this example, the respective laser beams R, G, and B are modulated in intensity at a given frequency and the on/off operation of the output is controlled according to a modulation signal and a pulse modulation signal, as will be described later.
The R light source 22r, the G light source 22g, and the B light source 22b oscillate and emits at predetermined wavelength bands in such a manner that the laser beams R, G, and B excite the fluorochromes so as to generate fluorescence having specific wavelength bands. The fluorochromes that are excited by the laser beams R, G, and B are adhered to the specimen 12 such as a biologic material or micro beads to be measured in fluorochrome, and when the specimen 12 passes through the tube 30 as the object to be measured, the fluorochromes generate fluorescence at specific wavelengths upon receiving the irradiation of the laser beams R, G, and B at the measurement point.
Fig. 3 is a diagram schematically showing the emitting wavelength of the laser beam, and the spectrum intensity distribution of fluorescence that is generated by the fluorochrome by using the laser beam. For example, three kinds of fluorescence consisting of fluorescence having a center wavelength of λ₁₂, fluorescence having a center wavelength of λ₁₃, and fluorescence having a center wavelength of λ₁₄ are generated by three different fluorochromes by irradiation of the laser beam having a wavelength λ₁₁ which is output from the B light source. Likewise, two kinds of fluorescence (A₂₂, λ₂₃) are generated by irradiation of the laser beam having a wavelength λ₂₁ that is output from the G light source. Further, one kind of fluorescence (λ₃₂) is generated by irradiation of the laser beam having a wavelength λ₃₁ that is output from the B light source.
The light receiving section 24 is so arranged as to face the laser light source section 22 with the tube 30 provided therebetween. The light receiving section 24 is equipped with a photoelectric converter that outputs a detection signal indicating that the specimen 12 passes by the measurement point when the laser beam is scattered forward by the specimen 12 that passes by the measurement point. The signal that is output from the light receiving section 24 is supplied to the control/processing section 28, and used as a trigger signal that announces a timing at which the specimen 12 is passing by the measurement point in the tube 30 in the control/processing section 28.
On the other hand, the light receiving section 26 is so arranged as to be perpendicular to the output direction of the laser beam that is output from the laser light source section 22 and also perpendicular to the moving direction of the specimen 12 in the tube 30. The light receiving section 26 is equipped with photoelectric converters that receives the fluorescence that is generated by the specimen 12 that has been irradiated at the measurement point.
Fig. 4 is a schematic structural diagram showing the configuration of an example of the light receiving section 26.
The light receiving section 26 shown in Fig. 4 includes a lens system 26a that focuses the fluorescence signal from the specimen 12, dichroic mirrors 26b₁ and 26b₂, band pass filters 26c₁ to 26c₃, and photoelectric converters 27a to 27c such as photoelectron multiple tubes.
The lens system 26a is so designed as to focus the fluorescence that has been input to the light receiving section 26 on the light receiving surface of the photoelectric converters 27a to 27c. The dichroic mirrors 26b₁ and 26b₂ are mirrors that reflect the fluorescence having given wavelength bands and transmit other fluorescence. The reflection wavelength bands and the transmission wavelength bands of the dichroic mirrors 26b₁ and 26b₂ are set so that through filtering the fluorescence by the band pass filters 26c₁ to 26c₃, the fluorescence having the predetermined wavelength bands are taken in by the photoelectric converters 27a to 27c.
The band pass filters 26c₁ to 26c₃ are filters that are disposed in front of the light receiving surfaces of the respective photoelectric converters 27a to 27c, and transmit only the fluorescence of the given wavelength bands. The wavelength bands of the transmitted fluorescence are set in correspondence with the wavelength band of the fluorescence that is generated by the fluorochromes shown in Fig. 2. For example, the wavelength band of the transmitted fluorescence is a band of a constant wavelength width centered on the wavelength λ₁₃ which is generated by the irradiation of the laser beam of the wavelength λ₁₁ which is generated from the B laser beam. In this case, as shown in Fig. 3, because the fluorescence centered on the wavelength λ₂₂ which is generated by irradiation of the laser beam having the wavelength λ₂₁ which has been output from the G light source has a center wavelength in the vicinity of the wavelength λ₁₃, the band pass filter transmits the above fluorescence together with the fluorescence centered on the wavelength λ₁₃. However, the fluorescence having the wavelength λ₂₁ as the center wavelength and the fluorescence having the wavelength λ₁₃ as the center wavelength are received by the photoelectric converters 27a to 27c as the fluorescence having signal information that has been modulated by a coding sequence signal (codes 1 to 3 of Fig. 7) which will be described later. In other words, signal processing that will be described later is conducted on the fluorescence signal that is received and generated, thereby making it possible to identify which laser beam generates the signal fluorescence.
Each of the photoelectric converters 27a to 27c is formed of, for example, a sensor having a photoelectron multiple tube, which converts a light that has been received by the photoelectric surface into an electric signal. In this example, since the received fluorescence is received as an optical signal having the signal information, the output electric signal is the fluorescence signal having the signal information. The fluorescence signal is amplified by an amplifier and then supplied to the control/processing section 28.
As shown in Fig. 5, the control/processing section 28 includes a signal generating section 40, a signal processing section 42, and a controller 44. The signal generating section 40 and the controller 44 constitute a light source control section that generates a modulation signal having a given frequency. The signal generating section 40 is a section that generates a modulation signal for modulating (amplitude modulating) the intensity of the laser beam at a given frequency. More specifically, the signal generating section 40 includes an oscillator 46, a power splitter 48, and amplifiers 50 and 52. The signal generating section 40 supplies the generated modulation signal to the power splitter 35 of the laser light source 22 and also to the signal processing section 42. The reason that the modulation signal is supplied to the signal processing section 42 is because the modulation signal is used as a reference signal for detecting the fluorescence signals that are output from the photoelectric converters 27a to 27c as described below. The modulation signal is a sine wave signal of a given frequency, and set to a frequency ranging from 10 to 100 MHz.
The signal processing section 42 is a section that extracts information related to the phase delay of the fluorescence that is generated from the micro beads by irradiation of the laser beam, by using the fluorescence signal that is output from the photoelectric converters 27a to 27c. The signal processing section 42 includes amplifiers 54a to 54c that amplify the fluorescence signals that are output from the photoelectric converters 27a to 27c, a power splitter 56 that splits a modulation signal that is a sine wave signal supplied from the signal generating section 40 to the respective amplified fluorescence signals, and IQ mixers 58a to 58c that mix the modulation signal as the reference signal with the amplified fluorescence signals.
The IQ mixers 58a to 58c are devices that mix the fluorescence signals that are supplied from the photoelectric converters 27a to 27c with the reference signal that is the modulation signal which is supplied from the signal processing section 40. More specifically, as shown in Fig. 6, each of the IQ mixers 58a to 58c multiplies the reference signal by the fluorescence signal (RF signal) to calculate a processing signal including the cos component and high frequency component of the fluorescence signal. Further, the IQ mixer multiplies a signal obtained by shifting the phase of the reference signal by 90 degrees by the fluorescence signal to calculate a processing signal including the sin component and the high frequency component of the fluorescence signal. The processing signal including the cos component and the processing signal including the sin component are supplied to the controller 44.
The controller 44 is a section that controls the signal generating section 40 so as to generate a sine wave signal having a given frequency. The controller 44 also controls the on/off operation of the output of the laser beam of the laser drivers 34r, 34g, and 34b in the laser light source section 22 by using the coding sequence signal. The controller 44 further removes the high frequency component from the processing signals including the cos component and the sin component of the fluorescence signal that has been obtained by the signal process section 42 to obtain the cos component and the sin component of the fluorescence signal.
More specifically, the controller 44 has a system controller 60, a low pass filter 62, an amplifier 64, and an A/D converter 66.
The system controller 60 gives instructions for controlling the operation of the respective sections, and manages the entire operation of the flow cytometer 10. The low pass filter 62 removes the high frequency component from the processing signals which are combinations of the high frequency component and the cos component, the sin component that have been calculated by the signal processing section 42. The amplifier 64 amplifies the processing signal of the cos component and the sin component from which the high frequency component has been removed. The A/D converter 66 samples the amplified processing signal. More specifically, the system controller 60 determines the oscillation frequency of the oscillator 46 in order to modify the intensity of the laser beam. In addition, the system controller 60 generates the pulse control signal that controls the on/off operation of the output of the laser beam. The pulse control signal is produced by one coding sequence signal that is selected from plural coding sequence signals that are orthogonal to each other. The coding sequence signal is constituted by signal values of one bit, and is coded by the bit number of a given code length.
Hereinafter, the coding sequence signal will be described.
The controller 44 generates the coding sequence signal that is a reference by using a sequence code C = {a₀, a₁, a₂, ..., a_N-1} (N is a natural number representative of a code length). The controller 44 also generates the coding sequence signal by using a sequence code T_q1·c (T_q1 is an operator that is shifted by q1 bit in a higher bit direction) which is obtained by shifting the sequence code C by q1 bits in the higher bit direction. In this example, the sequence code T_q1·C is {a_q1, a_q1+1, a_q1+2, ..., a_q1+N-1}. Further, the controller 44 generates the coding sequence signal by using the sequence code T_q2·C that is obtained by shifting the sequence code C by q2 bits (for example, q2 = 2 x q1) in the higher bit direction.
Since the sequence codes C, T_q1·C, T_q2·C which are used to generate the coding sequence signal have the characteristic orthogonal to each other, the generated coding sequence signals are also orthogonal to each other.
As an example of the sequence code C, as indicated below, there is a PN sequence (pseudorandom noise sequence) that is coded by using, for example, a coefficient h_j (j = an integer of 1 to 8) and an initial value a_k (k is an integer of 0 to 7). The PN sequence can be defined by, for example, the following formula (1). In the formula (1), the order is eighth order. In the formula, N is the code length of the sequence code, and for example, N = 255 (=2⁸-1) bits are set.
[EX. 1] $\begin{array}{l} h_{1} = 0, h_{2} = 1, h_{3} = 1, h_{4} = 1, h_{5} = 0, h_{6} = 0, h_{7} = 0, h_{8} = 1, \\ a_{0} = 0, a_{1} = 0, a_{2} = 0, a_{3} = 0, a_{4} = 0, a_{5} = 0, a_{6} = 0, a_{7} = 1 \\ \begin{matrix} a_{8 + i} = \sum_{j = 1}^{8} h_{j} a_{8 - j + i} & (i = 0, 1, 2, \dots) & (\mod 2) \end{matrix} \end{array}$
In the case where the sequence code C is the PN sequence code, since the code length is a cyclic code of N, aN = a₀, a_N+1 = a₁, .... Further, when another sequence code having the same code length as that of the sequence code C is set as C'={b0, b₁, b₂, ..., b_N-1}, and the above operator Tq is set as a sequence code T_q·C'={b_q, b_q+1, b_q+2' ..., b_q+N-1} acting on the sequence code C', a mutual correlation function R_cc' (q) between the sequence codes C and C' is defined as the following formula (2). In this formula, N_A is the number in which a term a_i and a term b_q+i (i is an integer that is equal to or more than 0 and equal to or less than N-1) in the sequence code coincide with each other, and N_D is the number in which the term a_i and the term b_q+i in the sequence code does not coincide with each other. Further, the sum of N_A and N_D is the code length N (N_A+N_D = N). In this formula, i and q+i are considered in mod (N).
[EX. 2] $R_{ccʹ} (q) = \frac{N_{A} - N_{D}}{N_{A} + N_{D}}$
A result of adding two sequence codes in mod (2) in each of the terms in the above PN sequence characteristically becomes a PN sequence obtained by cyclically shifting the original PN sequence, and the number in which the value of the PN sequence is zero is smaller by 1 than the number in which the value is 1, so that N_A-N_D = -1. As a result, the values represented by the following formulae (3) and (4) are expressed in the PN sequence.
[EX. 3] $R_{ccʹ} (q) = 1 \{q = 0 (modN)\}$

[EX. 4] $R_{ccʹ} (q) = - 1 / N \{q \neq 0 (modN)\}$
In the case where the bit shift quantity is 0, that is, q=0 in the above formula (3), a value of R_cc' (q) is 1 as represented by formula (3), and there is provided self-correlation. On the other hand, in the case where the bit shift quantity is not 0, that is, q >0, R_cc' (q) becomes - (1/N) as represented by formula (4). In this expression, when the code length N is increased, a value of R_cc'(q) (g>0) approaches 0. In other words, the sequence codes C and C' have self-correlativity and orthogonality.
The above sequence code having self-correlativity and orthogonality is used to generate the coding sequence signal consisting of binary values having a value of 0 and a value of 1.
Fig. 7A shows an example of the generated coding sequence signal. The coding sequence signal of a code 1 is a signal having a code length of N = 255 bits, and a product of the code length N and a time resolved width Δt is a time length from times 0 to t₃ of Fig. 7A. The on/off operation of output of the laser light source is intermittently controlled in such a manner that the laser beam is output when the value is 1, and the laser beam is not output when the value is 0 in the above signal.
In this example, in the coding sequence signal, a signal at a time 0 in a code 2 is generated in correspondence with a signal at a time t₁ in a code 1, and a signal after the time 0 in the code 2 is generated in correspondence with a signal after the time t₁ in the code 1. Likewise, in the coding sequence signal, a signal at the time 0 in a code 3 is generated in correspondence with a signal at a time t₂ (for example, t₂ = 2 x t₁) in the code 1, and a signal after the time 0 in the code 3 is generated in correspondence with a signal after the time t₂ in the code 1.
A light source control section 28a cyclically and repetitively generates those signals, and is configured so that the code 1 is supplied to the laser driver 34r, the code 2 is supplied to the laser driver 34g, and the code 3 is supplied to the laser driver 34b, as pulse control signals, respectively.
The coding sequence signals in the present invention are generated by using the sequence code of the above PN sequence. However, the generation of the coding sequence signal having self-correlativity and orthogonality in the present invention is not limited to the above method, and any methods can be applied so far as the coding sequence signal having self-correlativity and orthogonality is generated.
Fig. 7B shows an association between the pulse modulation due to the coding sequence signal and the intensity modulation due to the frequency of the laser beam. In the case where the laser beam is on due to the pulse modulation, the laser beam is so modulated as to vibrate in intensity in a cycle shorter than at least a time of the on-state.
The system controller 60 of the controller 44 generates the coding sequence signal by using the sequence code as shown in Fig. 7A, and supplies the coding sequence signal to the respective laser drivers 34r, 34g, and 34b as a pulse control signal that controls the on/off operation of output of the laser beam.
It is preferable that, in the sampling in the A/D converter 66 of the controller 44, the time resolution width (sampling interval) of sampling be made to correspond to the time resolution width of the coding sequence signal in order to efficiently calculate the correlation function between the coding sequence signal and the fluorescence signal as will be described below. For example, when the time resolution width of the coding sequence signal is 0.5 microsec, it is preferable that the time resolution width of sampling of the fluorescence signal be also set to 0.5 microsec or 1/integer of 0.5 microsec.
The analyzing device 80 is a device that obtains a phase delay angle with respect to the laser beam of fluorescence, also obtains a fluorescence relaxation time constant (= fluorescence relaxation time) from the phase delay angle, and specifies which laser beam's irradiation causes the fluorescence signal that has been output from the light receiving section 26. The analyzing device 80 is a processor section that is formed of a computer and calculates a fluorescence relaxation time (= fluorescence relaxation time constant)of the present invention.
The processing signal including the cos component and the sin component of the fluorescence signal includes also information on the coding sequence signal. Therefore, the analyzing device 80 first conducts coding identification conversion using self-correlativity and orthogonality of the processing signal, and extracts the values of the cos component and the sin component of the fluorescence signal in each of the laser beams. The analyzing device 80 obtains the phase delay angle with respect to the laser beam of fluorescence by using the cos component and the sin component. The analyzing device 80 obtains the fluorescence relaxation time constant (fluorescence relaxation time) according to the phase delay angle, and identifies the fluorochrome, thereby specifying the kind of specimen 12. Further, the analyzing device 80 is informed of the coding sequence signal used in the coding identification conversion, to thereby specify which laser beam's irradiation causes the fluorescence signal.
The obtained phase shift angle depends on the fluorescence relaxation time constant of fluorescence that is generated by the fluorochrome. In the case where the phase shift angle is represented by, for example, a first order lag relaxation process, the cos component and the sin component are expressed by the following formulae (5) and (6).
[EX. 5] $\cos (θ) = \frac{1}{\sqrt{1 + (ω τ)^{2}}}$

[EX. 6] $\sin (θ) = \frac{ω τ}{\sqrt{1 + (ω τ)^{2}}}$

In the expressions, θ is the phase shift angle, ω is the modulation frequency of the laser beam, and τ is the fluorescence relaxation time constant. When it is assumed that the initial fluorescence intensity is I₀ as shown in Fig. 8, the fluorescence relaxation time constant τ is a period of time from initial time point to a time point where the fluorescence intensity becomes I₀/e (e is a base of natural logarithm, e ≒ 2.71828).
The analyzing device 80 obtains the phase shift angle θ according to the ratio tan (θ) of the cos component and the sin component of the fluorescence signal, and can obtain the fluorescence relaxation time constant τ from the above fromulae (5) and (6) by using the phase shift angle θ. As described above, the fluorescence relaxation time constant τ depends on the kind of fluorochrome. Further, when two kinds of fluorochromes are mixed with each other at a different ratio, an apparent fluorescence relaxation time constant τ also changes according to the ratio. Thus, the analyzing device 80 obtains the fluorescence relaxation time constant τ, thereby enabling the ratio of those two fluorochromes to be specified.
As described above, the analyzing device 80 irradiates the fluorescence detection micro beads with the intensity-modulated laser beam, and detects the fluorescence that is generated at that time, thereby making it possible to identify the kind of generated fluorescence. As a result, it is possible to specify the kind of specimen 12 such as the micro beads.
A signal of fluorescence that is generated by the fluorochrome in the specimen 12 is a fluorescence signal that is caused by the laser beam that has been modulated (output is controlled) according to the known coding sequence signal which has been generated by the system controller 60. Thus, the fluorescence signal is also a signal whose optical intensity has been modulated according to the coding sequence signal that has been generated by the system controller 60. Accordingly, the analyzing device 80 synchronizes the code (coding sequence signal) that is an input signal generated by the system controller 60 with the fluorescence signal that is a response signal, and investigates the correlation function, thereby making it possible to determine which code has been used to modulate the laser beam that has caused the fluorescence signal included in the fluorescence signal. In other words, in the case where the analyzing device 80 calculates the correlation function between the code that has been generated by the system controller 60 and the fluorescence signal and the code is found to have a high correlation value with respect to the fluorescence signal, the fluorescence signal generated by the laser beam which has been modulated by the code (coding sequence signal) is included. On the other hand, in the case where the code is found to have extremely low or zero correlation, the fluorescence signal generated by the laser beam that has been modulated by the code is not included. Accordingly, the laser beam that has been modulated by the different code in each of the laser light sources is output, thereby making it possible to determine which laser beam causes the received fluorescence.
The analyzing device 80 repetitively averages the correlation function between the code that is an input signal and the fluorescence signal that is a response signal according to the cycle of circulating codes to obtain a stable value. The analyzing device 80 specifies which laser beam causes the received fluorescence. Further, since the wavelength bands of the laser beams that have been received by the respective photoelectric converters 27a to 27c are known, the analyzing device 80 is capable of specifying the kind of fluorescence.
As described above, the analyzing device 80 is capable of identifying and specifying by irradiation of which laser beam and in which wavelength band the fluorochrome in the specimen 12 includes the signal of fluorescence that has been radiated.
As described above, the analyzing device 80 specifies the fluorochrome disposed in the specimen 12 such as the micro beads by using the fluorescence relaxation time constant of fluorescence generated by the fluorochrome and information indicating which laser beam causes the fluorescence signal, thereby making it possible to specify the kind of specimen 12 that passes by the tube 30. In the case of the micro beads, since, for example, a given DNA fragment is provided on corresponding with the fluorochrome of the micro beads, the fluorochrome is specified, to thereby enable the kind of DNA fragment on the micro beads to be notified of. As a result, in the case where the analyzing device 30 measures the fluorescence of the micro beads and the fluorescence of the DNA fragment of an object to be detected at the same time, the analyzing device 30 determines that the DNA fragment of the object to be detected acts on a specific DNA fragment of the micro beads, to conduct the biological coupling therewith. In this way, the analyzing device 80 is capable of analyzing which micro beads the DNA fragment of the object to be detected is coupled with.
In the above manner, the analyzing device 80 obtains the histogram of the kind or the various characteristics of biologic material in the specimen 12 in a short time.
The flow cytometer 10 is configured as described above.
The signal processing device 20 of the flow cytometer 10 thus configured allows the signal of a given frequency to be generated in the oscillator 46 according to an instruction from the controller 44, the signal to be amplified by the amplifier 50, and the signal to be supplied to the laser light source section 22 and the signal processing section 42. In this state, the specimen 12 flows in the tube 30 to form a flow. The flow has, for example, a flow rate of 1 to 10 m/sec in the flow path diameter of 100 µm. Further, in a case where the micro beads are used as the specimen 12, the spherical diameter of the micro beads is several µm to 30 µm.
When the measurement point is irradiated with the laser beam, a detection signal that detects the pass of the specimen 12 by the light receiving section 24 is output to the controller 44 as a trigger signal.
The controller 44 deals with the detection signal as the trigger signal, and generates the coding sequence signal having self-correlativity and orthogonality to another coding sequence signal simultaniously with the trigger signal output. The coding sequence signal is cyclically and repetitively generated. The coding sequence signal is supplied to the laser drivers 34r, 34g, and 34b in order to be used as a pulse control signal that controls the on/off operation of the output of the laser beam from the laser light source section 22.
In the laser light source section 22, the on/off operation of the output of each laser beam is controlled according to the pulse control signal to generate the laser beam having the signal information of the pulse modulation according to the coding sequence signal. The laser beam is used to excite the fluorochrome in the specimen 12 which passes by the measurement point. The fluorochrome generates fluorescence by irradiation of the laser beam. The fluorescence generated by the fluorochrome is received by the light receiving section 26. The laser beam whose output is on is modulated in intensity at a given frequency.
The fluorescence from the fluorochrome which is generated by irradiation of the above laser beam is modulated in intensity at the given frequency with a phase delay angle, and the fluorescence that is excited and generated according to the on/off operation of the laser beam is also an on/off signal.
A period of time from the detection by the light receiving section 24 of the specimen 12 passing by to the irradiation of the modulated laser beam as described above is extremely short. The specimen 12 is irradiated with the laser beam whose on/off operation is controlled according to the coding sequence signal that is cyclically repeated while the laser beam is modified in the amplitude at the given frequency during several µsec to several tens µsec during which the specimen 12 passes by the measurement point.
In this example, the modulation frequency of the laser beam is, for example, 10 to 100 MHz. Further, in the case where the coding sequence signal is generated, for example, with the time resolution width of 1 µsec and when the code length N of the coding modulation signal is 7 bits, the coding sequence signal is repetitively and cyclically generated with 7 µsec (= 1.0 x 7) as one cycle. The laser beam is modulated on the basis of the coding sequence signal that is repetitively generated. Accordingly, the laser beam has the coding sequence signal with 7 µsec as one cycle circulated several times to several tens times during several µsec to several tens µsec.
The fluorescence signals that are received and output by the photoelectric converters 27a to 27c of the light receiving section 26 are amplified by the amplifiers 54a to 54c, and then mixed with the modulation signal that is a sine wave signal that has been supplied from the signal generating section 40 by means of the IQ mixers 58a to 58c.
The IQ mixers 58a to 58c generates a mixed signal resulting from multiplying the modulation signal (reference signal) that is the sine wave signal by the fluorescence signal. The IQ mixers 58a to 58c also generate a mixed signal resulting from multiplying the signal that is shifted in phase from the modulation signal (reference signal) that is the sine wave signal by 90 degrees by the fluorescence signal.
Subsequently, those generated two mixed signals are transmitted to the low pass filter 62 of the controller 44, and the high frequency components are removed from those mixed signals to extract the signal having the cos component and the sin component of the fluorescence signal. The signal having the cos component and the sin component of the fluorescence signal is amplified, subjected to A/D conversion, and then transmitted to the analyzing device 80. The A/D conversion is synchronized with a timing of the trigger signal from the light receiving section 24, and the fluorescence signal is sampled with the same time resolution width as the time resolution width Δt of the coding sequence signal. The sampling is, for example, a 16 bits sampling (sampling of the gradation of 0 to ±32767). The fluorescence signal is generated by the laser beam that has been modulated in pulse according to the coding sequence signal. Thus, the sampled data which is obtained from the fluorescence signal includes information on the coding sequence signal.
For example, the intensity amplitude A_i(t) of the time modulated laser beam that is output from the i-th (i= natural number of 1 to 3) laser light source section 22 is described as the following formulae (7) (p_i(t) is a time modulated component of the PN coding modulation signal, ω is a modulation frequency), and a reference signal (modulation signal) A₀(t) that is supplied to the IQ mixers 58a to 58c is described as the following formula (8). In this case, a signal A₉₀(t) that is shifted in phase from the modulation signal (reference signal) that is a sine wave signal by 90 degrees, which is used in each of the IQ mixers 58a to 58c, is described as the following formula (9). On the other hand, when the amplitude of the fluorescence signal is described according to the following formula (10), the mixed signals at the IQ mixers 58a to 58c are represented by the following formula (11). The higher order components of those two mixed signals are removed by using the low pass filter 62, and is then subjected to A/D conversion to produce digital signals.
[EX. 7] $\begin{array}{l} A_{i} (t) = p_{i} (t) \cdot \cos (ωt) \\ (i = 1, 2, 3) \end{array}$

[EX. 8] $A_{0} (t) = \cos (ωt)$

[EX. 9] $A_{90} (t) = - \sin (ω t)$

[EX. 10] $A (t) = \sum_{i = 1}^{3} p_{i} (t) \cdot r_{i} \cdot \cos (ω t + θ_{i})$

(r_i is the intensity amplitude of fluorescence)
[EX. 11] $mixed signal = {\begin{matrix} \frac{1}{2} \sum_{i = 1}^{3} p_{i} (t) \cdot r_{i} \cdot \cos θ_{i} + (higher order component) \\ \frac{1}{2} \sum_{i = 1}^{3} p_{i} (t) \cdot r_{i} \cdot \sin θ_{i} + (higher order component) \end{matrix}$
The coding sequence signal used in the time modulation of the laser beam has the self-correlation, and has orthogonality with respect to another coding sequence signal. Accordingly, a correlation between the coding sequence signal and the digital signal that has been subjected to A/D conversion is calculated to conduct coding identification conversion that decomposes the digital signal in each of the laser signals. That is, 1/2· r_i· cos(θ_i) and 1/2· r_i· sin(θ_i) in the formula (11) are obtained through the coding identification conversion. In this expression, θ_i represents the phase delay angle of the fluorescence that is generated by the laser beam which is output from the i-th laser light source section 22 with respect to the laser beam. Therefore, tan(θ_i) can be obtained by using the values of 1/2· r_i· cos (θ_i) and 1/2· r_i sin(θ_i). The fluorescence relaxation time constant τ is obtained by using the value of tan(θ_i) and the above-mentioned formulae (5) and (6).
The fluorescence relaxation time constant τ depends on the fluorochrome, therefore by using a different kind of fluorochrome for a different kind of specimen 12 such as the micro beads (kind of biologic material adhered to the specimen 12 is different), the kind of fluorochrome can be specified. As a result, the kind of specimen 12 can be specified. Accordingly, the kind of specimen 12 is specified by the fluorescence that is generated by the specimen 12. In the case of additionally detecting the fluorescence that is generated by the biologic material having a given fluorochrome, it is possible to specify which kind of specimen 12's structure the biologic material is biologically coupled with. The analysis can be conducted by using those results.
In particular, when two different kinds of fluorochromes are mixed together at different ratios, the apparent fluorescence relaxation time constant τ also changes according to the ratio. Thus, the mixture ratio is changed, thereby making it possible to set an extremely large number of fluorescence relaxation time constants. Accordingly, it is possible to set a variety of micro beads which are capable of identifying the generated fluorescence by using the fluorochromes having different fluorescence relaxation time constants for each kind of the micro beads.
In addition, since the coding sequence signal having the self-correlation can be given in conducting the above coding identification conversion, it is possible to specify which laser beam causes the fluorescence signal. In the conventional art, since the laser beam is not modulated and the modulated signal information is not included in the fluorescence, it is impossible to specify which laser beam excites the fluorescence.
In the present invention, even if the fluorescence is emitted at the same wavelength, the coding sequence signal of the laser beam used for excitation is changed, thereby making it possible to receive the fluorescence as a different fluorescence signal. As a result, it is possible to specify a large number of laser beams that are used for excitation of the fluorochrome in a short time by using a large number of coding sequence signals having orthogonality. Accordingly, even if there are a large number of fluorochromes that are close to each other in the wavelength band of fluorescence, or even if a large number of laser beams are bundled together and irradiated at once, the signal information of the irradiated laser beam is included in the fluorescence. It is possible to specify the fluorochrome that is adhered to the specimen so far as the signal information in the light receiving signal can be identified.
As described above, according to the present invention, the fluorescence relaxation time constant of fluorescence which is generated by the fluorochrome is calculated, thereby making it possible to increase the kinds of identifiable fluorochromes. In particular, two kinds of fluorochromes that are different in the fluorescence relaxation time constant are mixed together at a predetermined ratio, thereby making it possible to provide a fluorescence relaxation time constant that is different from two kinds of fluorescence relaxation time constants. As a result, the number of the identifiable fluorochromes are significantly increased. As examples of two kinds of fluorochromes, it is preferable that one kind be a fluorochrome that is selected from a group consisting of Cascade Blue, Cascade Yellow, Alexa Fluor 405, DAPI, Dapoxyl, Dialkylaminocoumarin, Hydroxycoumarin, Marine Blue, Pacific Blue, and PyMPO. Another fluorochrome is preferably a semiconductor quantum fluorochrome that is selected from a group consisting of Q-Dot (product name of Quantum Dot Company) and Evic-Tag (product name of Evident Technology Co.). The former fluorochrome is shorter in the fluorescence relaxation time constant than the latter fluorochrome. The ratio at which those two kinds of fluorochromes are mixed together is changed, thereby making it possible to greatly change the fluorescence relaxation time constant. For example, Q-Dot has the fluorescence relaxation time constant of 20 to 40 nanoseconds.

Claims

A fluorescence detecting device using an intensity-modulated laser beam, which irradiates an object to be measured with a laser beam to receive fluorescence generated by the object to be measured, and carries out signal processing of a fluorescence signal obtained when receiving the fluorescence, comprising:
a laser light source section that outputs the laser beam with which the object to be measured is irradiated;

a light receiving section that outputs the fluorescence signal of the fluorescence generated by the object to be measured which is irradiated with the laser beam;

a light source control section that generates a modulation signal having a given frequency in order to time-modulate an intensity of the laser beam that is output from the laser light source section; and

a processing section that calculates, by using the modulation signal, a fluorescence relaxation time of the fluorescence of the object to be measured based on the fluorescence signal that is output from the light receiving section by irradiating the object to be measured with the time-modulated laser beam,

the device being characterizing that the light source control section uses as a pulse control signal a coding sequence signal that is selected from a plurality of coding sequence signals which have signal values of one bit coded with a given length and are orthogonal to each other, and sets and controls on/off of outpour of the laser beam so that an on-time of the output of the laser beam from the laser light source section is longer than one cycle time of the time modulation of the laser beam from the laser light source section is longer than once cycle time of the time modulation of the laser beam, and the processing section calculates the fluorescence relaxation time and identifies the fluorescence from the object to be measured, by using the coding sequence signal based on a light receiving signal that is output from the light receiving section.
The fluorescence detecting device using an intensity-modulated laser beam according to claim 1, wherein the processing section obtains a phase delay with respect to the modulation signal of the fluorescence signal to calculate the fluorescence relaxation time.
The fluorescence detecting device using an intensity-modulated laser beam according to claim 1 or 2.
wherein the plurality of coding sequence signals are configured by shifting one coding sequence signal in a bit direction and the coding sequence signals become orthogonal to each other by the shifting.
The fluorescence detecting device using an intensity-modulated laser beam according to any one of claims 1, 2 or 3,
wherein the laser light source section includes a plurality of laser light sources that output a plurality of laser beams,
wherein the light source control section controls the on/off of outputs of the laser beams from the plurality of laser light sources by using the plurality of coding sequence signals that are orthogonal to each other, and
wherein the processing section separates each of fluorescence signals of the fluorescence that is generated by the object to be measured by irradiation of the respective laser beams, from the fluorescence signals which are overlapped together and outputted from the light receiving section, including optical signals of the plurality of laser beams, by using the coding sequence signals used for the outputs of the laser beams.